Medical devices containing rapamycin analogs

ABSTRACT

A medical device comprising a supporting structure having a coating on the surface thereof, the coating containing a therapeutic substance, such as, for example, a drug. Supporting structures for the medical devices that are suitable for use in this invention include, but are not limited to, coronary stents, peripheral stents, catheters, arterio-venous grafts, by-pass grafts, and drug delivery balloons used in the vasculature. Drugs that are suitable for use in this invention include, but are not limited to,

[0001] This application is a continuation-in-part of U.S. Ser. No.09/433,001, filed Nov. 2, 1999, which is a divisional of U.S. Ser. No.09/159,945, filed Sep. 24, 1998, now U.S. Pat. No. 6,015,815,incorporated herein by reference.

TECHNICAL FIELD

[0002] The present invention relates to novel chemical compounds havingimmunomodulatory activity and synthetic intermediates useful for thepreparation of the novel compounds, and in particular to macrolideimmunomodulators. More particularly, the invention relates tosemisynthetic analogs of rapamycin, means for their preparation,pharmaceutical compositions containing such compounds, and methods oftreatment employing the same.

BACKGROUND OF THE INVENTION

[0003] The compound cyclosporine (cyclosporin A) has found wide usesince its introduction in the fields of organ transplantation andimmunomodulation, and has brought about a significant increase in thesuccess rate for transplantation procedures. Recently, several classesof macrocyclic compounds having potent immunomodulatory activity havebeen discovered. Okuhara et al., in European Patent ApplicationNo.184,162, published Jun. 11, 1986, disclose a number of macrocycliccompounds isolated from the genus Streptomyces, including theimmunosuppressant FK-506, a 23-membered macrocyclic lactone, which wasisolated from a strain of S. tsukubaensis.

[0004] Other related natural products, such as FR-900520 and FR-900523,which differ from FK-506 in their alkyl substituent at C-21, have beenisolated from S. hygroscopicus yakushimnaensis. Another analog,FR-900525, produced by S. tsukubaensis, differs from FK-506 in thereplacement of a pipecolic acid moiety with a proline group.Unsatisfactory side-effects associated with cyclosporine and FK-506 suchas nephrotoxicity, have led to a continued search for immunosuppressantcompounds having improved efficacy and safety, including animmunosupressive agent which is effective topically, but ineffectivesystemically (U.S. Pat. No. 5,457,111).

[0005] Rapamycin is a macrocyclic triene antibiotic produced byStreptomyces hygroscopicus, which was found to have antifungal activity,particularly against Candida albicans, both in vitro and in vivo (C.Vezina et al., J. Antibiot. 1975, 28, 721; S. N. Sehgal et al., J.Antibiot. 1975, 28, 727; H. A. Baker et al., J. Antibiot. 1978, 31, 539;U.S. Pat. No. 3,929,992; and U.S. Pat. No. 3,993,749).

[0006] Rapamycin alone (U.S. Pat. No. 4,885,171) or in combination withpicibanil (U.S. Pat. No. 4,401,653) has been shown to have antitumoractivity. In 1977, rapamycin was also shown to be effective as animmunosuppressant in the experimental allergic encephalomyelitis model,a model for multiple sclerosis; in the adjuvant arthritis model, a modelfor rheumatoid arthritis; and was shown to effectively inhibit theformation of IgE-like antibodies (R. Martel et al., Can. J. Physiol.Pharmacol., 1977, 55, 48).

[0007] The immunosuppressive effects of rapamycin have also beendisclosed in FASEB, 1989, 3, 3411 as has its ability to prolong survivaltime of organ grafts in histoincompatible rodents (R. Morris, Med. Sci.Res., 1989, 17, 877). The ability of rapamycin to inhibit T-cellactivation was disclosed by M. Strauch (FASEB, 1989, 3, 3411). These andother biological effects of rapamycin are reviewed in TransplantationReviews, 1992, 6, 39-87.

[0008] Rapamycin has been shown to reduce neointimal proliferation inanimal models, and to reduce the rate of restenosis in humans. Evidencehas been published showing that rapamycin also exhibits ananti-inflammatory effect, a characteristic which supported its selectionas an agent for the treatment of rheumatoid arthritis. Because both cellproliferation and inflammation are thought to be causative factors inthe formation of restenotic lesions after balloon angioplasty and stentplacement, rapamycin and analogs thereof have been proposed for theprevention of restenosis.

[0009] Mono-ester and di-ester derivatives of rapamycin (esterificationat positions 31 and 42) have been shown to be useful as antifungalagents (U.S. Pat. No. 4,316,885) and as water soluble prodrugs ofrapamycin (U.S. Pat. No. 4,650,803).

[0010] Fermentation and purification of rapamycin and 30-demethoxyrapamycin have been described in the literature (C. Vezina et al. J.Antibiot. (Tokyo), 1975, 28 (10), 721; S. N. Sehgal et al., J. Antibiot.(Tokyo), 1975, 28(10), 727; 1983, 36(4), 351; N. L. Pavia etal., J.Natural Products, 1991, 54(1), 167-177).

[0011] Numerous chemical modifications of rapamycin have been attempted.These include the preparation of mono- and di-ester derivatives ofrapamycin (WO 92/05179), 27-oximes of rapamycin (EPO 467606); 42-oxoanalog of rapamycin (U.S. Pat. No. 5,023,262); bicyclic rapamycins (U.S.Pat. No. 5,120,725); rapamycin dimers (U.S. Pat. No. 5,120,727); silylethers of rapamycin (U.S. Pat. No. 5,120,842); and arylsulfonates andsulfamates (U.S. Patent No. 5,177,203). Rapamycin was recentlysynthesized in its naturally occuring enantiomeric form (K. C. Nicolaouet al., J. Am. Chem. Soc., 1993, 115, 4419-4420; S. L. Schreiber, J. Am.Chem. Soc., 1993, 115, 7906-7907; S. J. Danishefsky, J. Am. Chem. Soc.,1993, 115, 9345-9346.

[0012] It has been known that rapamycin, like FK-506, binds to FKBP-12(Siekierka, J. J.; Hung, S. H. Y.; Poe, M.; Lin, C. S.; Sigal, N. H.Nature, 1989, 341, 755-757; Harding, M. W.; Galat, A.; Uehling, D. E.;Schreiber, S. L. Nature 1989, 341, 758-760; Dumont, F. J.; Melino, M.R.; Staruch, M. J.; Koprak, S. L.; Fischer, P. A.; Sigal, N. H. J.Immunol. 1990, 144,1418-1424; Bierer, B. E.; Schreiber, S. L.; Burakoff,S. J. Eur. J. Immunol. 1991, 21, 439-445; Fretz, H.; Albers, M. W.;Galat, A.; Standaert, R. F.; Lane, W. S.; Burakoff, S. J.; Bierer, B.E.; Schreiber, S. L. J. Am. Chem. Soc. 1991, 113, 1409-1411). Recentlyit has been discovered that the rapamycin/FKBP-12 complex binds to yetanother protein, which is distinct from calcineurin, the protein thatthe FK-506/FKBP-12 complex inhibits (Brown, E. J.; Albers, M. W.; Shin,T. B.; Ichikawa, K.; Keith, C. T.; Lane, W. S.; Schreiber, S. L. Nature1994, 369, 756-758; Sabatini, D. M.; Erdjument-Bromage, H.; Lui, M.;Tempest, P.; Snyder, S. H. Cell, 1994, 78, 35-43).

[0013] Percutaneous transluminal coronary angioplasty (PTCA) wasdeveloped by Andreas Gruntzig in the 1970's. The first canine coronarydilation was performed on Sep. 24, 1975; studies showing the use of PTCAwere presented at the annual meetings of the American Heart Associationthe following year. Shortly thereafter, the first human patient wasstudied in Zurich, Switzerland, followed by the first American humanpatients in San Francisco and New York. While this procedure changed thepractice of interventional cardiology with respect to treatment ofpatients with obstructive coronary artery disease, the procedure did notprovide long-term solutions. Patients received only temporary abatementof the chest pain associated with vascular occlusion; repeat procedureswere often necessary. It was determined that the existence of restenoticlesions severely limited the usefulness of the new procedure. In thelate 1980's, stents were introduced to maintain vessel patency afterangioplasty. Stenting is involved in 90% of angioplasty performed today.Before the introduction of stents, the rate of restenosis ranged from30% to 50% of the patients who were treated with balloon angioplasty.The recurrence rate after dilatation of in-stent restenosis may be ashigh as 70% in selected patient subsets, while the angiographicrestenosis rate in de novo stent placement is about 20%. Placement ofthe stent reduced the restenosis rate to 15% to 20%. This percentagelikely represents the best results obtainable with purely mechanicalstenting. The restenosis lesion is caused primarily by neointimalhyperplasia, which is distinctly different from atherosclerotic diseaseboth in time-course and in histopathologic appearance. Restenosis is ahealing process of damaged coronary arterial walls, with neointimaltissue impinging significantly on the vessel lumen. Vascularbrachytherapy appears to be efficacious against in-stent restenosislesions. Radiation, however, has limitations of practicality andexpense, and lingering questions about safety and durability.

[0014] Accordingly, it is desired to reduce the rate of restenosis by atleast 50% of its current level. It is for this reason that a majoreffort is underway by the interventional device community to fabricateand evaluate drug-eluting stents. Such devices could have manyadvantages if they were successful, principally since such systems wouldneed no auxiliary therapies, either in the form of peri-proceduraltechniques or chronic oral pharmacotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows blood concentrations ±SEM (n=3) oftatrazole-containing rapamycin analogs dosed in monkey.

[0016]FIG. 2 is a side view in elevation showing a stent suitable foruse in this invention.

[0017]FIG. 3A is a cross-sectional view of a vessel segment in which wasplaced a stent coated with a polymer only.

[0018]FIG. 3B is a cross-sectional view of a vessel segment in which wasplaced a stent coated with a polymer plus drug.

SUMMARY OF THE INVENTION

[0019] In one aspect of the present invention are disclosed compoundsrepresented by the structural formula:

[0020] or a pharmaceutically acceptable salt or prodrug thereof.

[0021] Another object of the present invention is to provide syntheticprocesses for the preparation of such compounds from starting materialsobtained by fermentation, as well as chemical intermediates useful insuch synthetic processes.

[0022] A further object of the invention is to provide pharmaceuticalcompositions containing, as an active ingredient, at least one of theabove compounds.

[0023] Yet another object of the invention is to provide a method oftreating a variety of disease states, including restenosis,post-transplant tissue rejection, immune and autoimmune dysfunction,fungal growth, and cancer.

[0024] In another aspect this invention provides a medical devicecomprising a supporting structure having a coating on the surfacethereof, the coating containing a therapeutic substance, such as, forexample, a drug. Supporting structures for the medical devices that aresuitable for use in this invention include, but are not limited to,coronary stents, peripheral stents, catheters, arterio-venous grafts,by-pass grafts, and drug delivery balloons used in the vasculature.Drugs that are suitable for use in this invention include, but are notlimited to,

[0025] or a pharmaceutically acceptable salt or prodrug thereof, whichincludes

[0026] or a pharmaceutically acceptable salt or prodrug thereof,(hereinafter alternatively referred to as A-179578), and

[0027] or a pharmaceutically acceptable salt or prodrug thereof;

[0028] or a pharmaceutically acceptable salt or prodrug thereof,(hereinafter alternatively referred to as SDZ RAD or40—O-(2-hydroxyethyl)-rapamycin);

[0029] or a pharmaceutically acceptable salt or prodrug thereof,(hereinafeter alternatively referred to as A-94507).

[0030] Coatings that are suitable for use in this invention include, butare not limited to, polymeric coatings that can comprise any polymericmaterial in which the therapeutic agent, i.e., the drug, issubstantially soluble. The coating can be hydrophilic, hydrophobic,biodegradable, or non-biodegradable. This medical device reducesrestenosis in vasculature. The direct coronary delivery of a drug suchas A-179578 is expected to reduce the rate of restenosis to a level ofabout 0% to 25%.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Definition of Terms

[0032] The term “prodrug,” as used herein, refers to compounds which arerapidly transformed in vivo to the parent compound of the above formula,for example, by hydrolysis in blood. A thorough discussion is providedby T. Higuchi and V. Stella, “Pro-drugs as Novel Delivery systems,” Vol.14 of the A. C. S. Symposium Series, and in Edward B. Roche, ed.,“Bioreversible Carriers in Drug Design,” American PharmaceuticalAssociation and Pergamon Press, 1987, both of which are incorporatedherein by reference.

[0033] The term “pharmaceutically acceptable prodrugs”, as used herein,refers to those prodrugs of the compounds of the present invention whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and lower mammals without unduetoxicity, irritation, and allergic response, are commensurate with areasonable benefit/risk ratio, and are effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe invention. Particularly preferred pharmaceutically acceptableprodrugs of this invention are prodrug esters of the C-31 hydroxyl groupof compounds of this invention.

[0034] The term “prodrug esters,” as used herein, refers to any ofseveral ester-forming groups that are hydrolyzed under physiologicalconditions. Examples of prodrug ester groups include acetyl, ethanoyl,pivaloyl, pivaloyloxymethyl, acetoxymethyl, phthalidyl, methoxymethyl,indanyl, and the like, as well as ester groups derived from the couplingof naturally or unnaturally-occurring amino acids to the C-31 hydroxylgroup of compounds of this invention.

[0035] The term “supporting structure” means a framework that is capableof containing or supporting a pharmaceutically acceptable carrier orexcipient, which carrier or excipient may contain a therapeutic agent,e.g., a drug or another compound. The supporting structure is typicallyformed of metal or a polymeric material.

Embodiments

[0036] In one embodiment of the invention is a compound of formula

[0037] In another embodiment of the invention is a compound of formula

Preparation of Compounds of this Invention

[0038] The compounds and processes of the present invention will bebetter understood in connection with the following synthetic schemeswhich illustrate the methods by which the compounds of the invention maybe prepared.

[0039] The compounds of this invention may be prepared by a variety ofsynthetic routes. A representative procedure is shown in Scheme 1.

[0040] As shown in Scheme 1, conversion of the C-42 hydroxyl ofrapamycin to a trifluoromethanesulfonate or fluorosulfonate leavinggroup provided A. Displacement of the leaving group with tetrazole inthe presence of a hindered, non-nucleophilic base, such as 2,6-lutidine,or, preferably, diisopropylethyl amine provided epimers B and C, whichwere separated and purified by flash column chromatography.

Synthetic Methods

[0041] The foregoing may be better understood by reference to thefollowing examples which illustrate the methods by which the compoundsof the invention may be prepared and are not intended to limit the scopeof the invention as defined in the appended claims.

EXAMPLE 1 42-Epi-(tetrazolyl)-rapamycin (less polar isomer) Example 1A

[0042] A solution of rapamycin (100 mg, 0.11 mmol) in dichloromethane(0.6 mL) at −78° C. under a nitrogen atmosphere was treated sequentiallywith 2,6-lutidine (53 uL, 0.46 mmol, 4.3 eq.) andtrifluoromethanesulfonic anhydride (37 uL, 0.22 mmol), and stirredthereafter for 15 minutes, warmed to room temperature and eluted througha pad of silica gel (6 mL) with diethyl ether. Fractions containing thetriflate were pooled and concentrated to provide the designated compoundas an amber foam.

Example 1 B 42-Epi-(tetrazolyl)-rapamycin (less polar isomer)

[0043] A solution of Example 1A in isopropyl acetate (0.3 mL) wastreated sequentially with diisopropylethylamine (87 L, 0.5 mmol) and1H-tetrazole (35 mg, 0.5 mmol), and thereafter stirred for 18 hours.This mixture was partitioned between water (10 mL) and ether (10 mL).The organics were washed with brine (10 mL) and dried (Na₂SO₄).Concentration of the organics provided a sticky yellow solid which waspurified by chromatography on silica gel (3.5 g, 70-230 mesh) elutingwith hexane (10 mL), hexane:ether (4:1 (10 mL), 3:1 (10 mL), 2:1 (10mL), 1:1 (10 mL)), ether (30 mL), hexane:acetone (1:1 (3OmL)). One ofthe isomers was collected in the ether fractions. MS (ESI) m/e 966 (M)⁻;

EXAMPLE 2 42-Epi-(tetrazolyl)-rapamycin (more polar isomer) Example 2A42-Epi-(tetrazolyl)-rapamycin (more polar isomer)

[0044] Collection of the slower moving band from the chromatographycolumn using the hexane:acetone (1:1) mobile phase in Example 1Bprovided the designated compound. MS (ESI) m/e 966 (M)⁻.

[0045] In vitro Assay of Biological Activity

[0046] The immunosuppressant activity of the compounds of the presentinvention was compared to rapamycin and two rapamycin analogs:40-epi-N-[2′-pyridone]-rapamycin and 40-epi-N-[4′-pyridone]-rapamycin,both disclosed in U.S. Pat. No. 5,527,907. The activity was deteminedusing the human mixed lymphocyte reaction (MLR) assay described by Kino,T. et al. in Transplantation Proceedings, XIX(5):36-39, Suppl. 6 (1987).The results of the assay demonstrate that the compounds of the inventionare effective immunomodulators at nanomolar concentrations, as shown inTable 1. TABLE 1 Human MLR Example IC₅₀ ± S.E.M. (nM) Rapamycin  0.91 ±0.36 2-pyridone 12.39 ± 5.3 4-pyridone  0.43 ± 0.20 Example 1  1.70 ±0.48 Example 2  0.66 ± 0.19

[0047] The pharmacokinetic behaviors of Example I and Example 2 werecharacterized following a single 2.5 mg/kg intravenous dose incynomolgus monkey (n=3 per group). Each compound was prepared as 2.5mg/mL solution in a 20% ethanol:30% propylene glycol:2% cremophor EL:48%dextrose 5% in water vehicle. The 1 mL/kg intravenous dose wasadministered as a slow bolus (˜1-2 minutes) in a saphenous vein of themonkeys. Blood samples were obtained from a femoral artery or vein ofeach animal prior to dosing and 0.1 (IV only), 0.25, 0.5, 1, 1.5, 2, 4,6, 9, 12, 24, and 30 hours after dosing. The EDTA preserved samples werethoroughly mixed and extracted for subsequent analysis.

[0048] An aliquot of blood (1.0 mL) was hemolyzed with 20% methanol inwater (0.5 ml) containing an internal standard. The hemolyzed sampleswere extracted with a mixture of ethyl acetate and hexane (1:1 (v/v),6.0 mL). The organic layer was evaporated to dryness with a stream ofnitrogen at room temperature. Samples were reconstituted in methanol:water (1:1, 150 μL). The title compounds (50 μL injection) wereseparated from contaminants using reverse phase HPLC with UV detection.Samples were kept cool (4° C.) through the run. All samples from eachstudy were analyzed as a single batch on the HPLC.

[0049] Area under the curve (AUC) measurements of Example 1, Example 2and the internal standard were determined using the Sciex MacQuan™software. Calibration curves were derived from peak area ratio (parentdrug/internal standard) of the spiked blood standards using leastsquares linear regression of the ratio versus the theoreticalconcentration. The methods were linear for both compounds over the rangeof the standard curve (correlation >0.99) with an estimated quantitationlimit of 0.1 ng/mL. The maximum blood concentration (CMAX) and the timeto reach the maximum blood concentration (TMAX) were read directly fromthe observed blood concentration-time data. The blood concentration datawere submitted to multi-exponential curve fifting using CSTRIP to obtainestimates of pharmacokinetic parameters. The estimated parameters werefurther defined using NONLIN84. The area under the bloodconcentration-time curve from 0 to t hours (last measurable bloodconcentration time point) after dosing (AUC_(0-t)) was calculated usingthe linear trapeziodal rule for the blood-time profiles. The residualarea extrapolated to infinity, determined as the final measured bloodconcentration (C_(t)) divided by the terminal elimination rate constant(β), and added to AUC_(0-t) to produce the total area under the curve(AUC_(0-t)).

[0050] As shown in FIG. 1 and Table 2, both Example 1 and Example 2 hada suprisingly substantially shorter terminal elimination half-life(t_(½)) when compared to rapamycin. Thus, only the compounds of theinvention provide both sufficient efficacy (Table 1) and a shorterterminal half-life (Table 2). TABLE 2 AUC t_(1/2) Compound ng · hr/mL(hours) Rapamycin 6.87 16.7 2-pyridone 2.55 2.8 4-pyridone 5.59 13.3Example 1 2.35 5.0 Example 2 2.38 6.9

[0051] Methods of Treatment

[0052] The compounds of the invention, including but not limited tothose specified in the examples, possess immunomodulatory activity inmammals (especially humans). As immunosuppressants, the compounds of thepresent invention are useful for the treatment and prevention ofimmune-mediated diseases such as the resistance by transplantation oforgans or tissue such as heart, kidney, liver, medulla ossium, skin,cornea, lung, pancreas, intestinum tenue, limb, muscle, nerves,duodenum, small-bowel, pancreatic-islet-cell, and the like;graft-versus-host diseases brought about by medulla ossiumtransplantation; autoimmune diseases such as rheumatoid arthritis,systemic lupus erythematosus, Hashimoto's thyroiditis, multiplesclerosis, myasthenia gravis, type I diabetes, uveitis, allergicencephalomyelitis, glomerulonephritis, and the like. Further usesinclude the treatment and prophylaxis of inflammatory andhyperproliferative skin diseases and cutaneous manifestations ofimmunologically-mediated illnesses, such as psoriasis, atopicdermatitis, contact dermatitis and further eczematous dermatitises,seborrhoeis dermatitis, lichen planus, pemphigus, bullous pemphigoid,epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas,cutaneous eosinophilias, lupus erythematosus, acne and alopecia areata;various eye diseases (autoimmune and otherwise) such askeratoconjunctivitis, vernal conjunctivitis, uveitis associated withBehcet's disease, keratitis, herpetic keratitis, conical cornea,dystrophia epithelialis corneae, corneal leukoma, and ocular pemphigus.In addition reversible obstructive airway disease, which includesconditions such as asthma (for example, bronchial asthma, allergicasthma, intrinsic asthma, extrinsic asthma and dust asthma),particularly chronic or inveterate asthma (for example, late asthma andairway hyper-responsiveness), bronchitis, allergic rhinitis, and thelike are targeted by compounds of this invention. Inflammation of mucosaand blood vessels such as gastric ulcers, vascular damage caused byischemic diseases and thrombosis. Moreover, hyperproliferative vasculardiseases such as intimal smooth muscle cell hyperplasia, restenosis andvascular occlusion, particularly following biologically- ormechanically- mediated vascular injury, could be treated or prevented bythe compounds of the invention. The compounds or drugs described hereincan be applied to stents that have been coated with a polymericcompound. Incorporation of the compound or drug into the polymericcoating of the stent can be carried out by dipping the polymer-coatedstent into a solution containing the compound or drug for a sufficientperiod of time (such as, for example, five minutes) and then drying thecoated stent, preferably by means of air drying for a sufficient periodof time (such as, for example, 30 minutes). The polymer-coated stentcontaining the compound or drug can then be delivered to the coronaryvessel by deployment from a balloon catheter. In addition to stents,other devices that can be used to introduce the drugs of this inventionto the vasculature include, but are not limited to grafts, catheters,and balloons. In addition, other compounds or drugs that can be used inlieu of the drugs of this invention include, but are not limited to,A-94507 and SDZ RAD). When used in the present invention, the coatingcan comprise any polymeric material in which the therapeutic agent,i.e., the drug, is substantially soluble. The purpose of the coating isto serve as a controlled release vehicle for the therapeutic agent or asa reservoir for a therapeutic agent to be delivered at the site of alesion. The coating can be polymeric and can further be hydrophilic,hydrophobic, biodegradable, or non-biodegradable. The material for thepolymeric coating can be selected from the group consisting ofpolycarboxylic acids, cellulosic polymers, gelatin,polyvinylpyrrolidone, maleic anhydride polymers, polyamides, polyvinylalcohols, polyethylene oxides, glycosaminoglycans, polysaccharides,polyesters, polyurethanes, silicones, polyorthoesters, polyanhydrides,polycarbonates, polypropylenes, polylactic acids, polyglycolic acids,polycaprolactones, polyhydroxybutyrate valerates, polyacrylamides,polyethers, and mixtures and copolymers of the foregoing. Coatingsprepared from polymeric dispersions such as polyurethane dispersions(BAYHYDROL, etc.) and acrylic acid latex dispersions can also be usedwith the therapeutic agents of the present invention.

[0053] Biodegradable polymers that can be used in this invention includepolymers such as poly(L-lactic acid), poly(DL-lactic acid),polycaprolactone, poly(hydroxy butyrate), polyglycolide,poly(diaxanone), poly(hydroxy valerate), polyorthoester; copolymers suchas poly (lactide-co-glycolide), polyhydroxy(butyrate-co-valerate),polyglycolide-co-trimethylene carbonate; polyanhydrides;polyphosphoester; polyphosphoester-urethane; polyamino acids;polycyanoacrylates; biomolecules such as fibrin, fibrinogen, cellulose,starch, collagen and hyaluronic acid; and mixtures of the foregoing.Biostable materials that are suitable for use in this invention includepolymers such as polyurethane, silicones, polyesters, polyolefins,polyamides, polycaprolactam, polyimide, polyvinyl chloride, polyvinylmethyl ether, polyvinyl alcohol, acrylic polymers and copolymers,polyacrylonitrile, polystyrene copolymers of vinyl monomers with olefins(such as styrene acrylonitrile copolymers, ethylene methyl methacrylatecopolymers, ethylene vinyl acetate), polyethers, rayons, cellulosics(such as cellulose acetate, cellulose nitrate, cellulose propionate,etc.), parylene and derivatives thereof; and mixtures and copolymers ofthe foregoing.

[0054] Another polymer that can be used in this invention ispoly(MPC_(w):LAM_(x):HPMA_(y):TSMA_(z)) where w, x, y, and z representthe molar ratios of monomers used in the feed for preparing the polymerand MPC represents the unit 2-methacryoyloxyethylphosphorylcholine, LMArepresents the unit lauryl methacrylate, HPMA represents the unit2-hydroxypropyl methacrylate, and TSMA represents the unit3-trimethoxysilylpropyl methacrylate. The drug-impregnated stent can beused to maintain patency of a coronary artery previously occluded bythrombus and/or atherosclerotic plaque. The delivery of ananti-proliferative agent reduces the rate of in-stent restenosis.

[0055] Other treatable conditions include but are not limited toischemic bowel diseases, inflammatory bowel diseases, necrotizingenterocolitis, intestinal inflammations/allergies such as Coeliacdiseases, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn'sdisease and ulcerative colitis; nervous diseases such as multiplemyositis, Guillain-Barre syndrome, Meniere's disease, polyneuritis,multiple neuritis, mononeuritis and radiculopathy; endocrine diseasessuch as hyperthyroidism and Basedow's disease; hematic diseases such aspure red cell aplasia, aplastic anemia, hypoplastic anemia, idiopathicthrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis,pernicious anemia, megaloblastic anemia and anerythroplasia; bonediseases such as osteoporosis; respiratory diseases such as sarcoidosis,fibroid lung and idiopathic interstitial pneumonia; skin disease such asdermatomyositis, leukoderma vulgaris, ichthyosis vulgaris, photoallergicsensitivity and cutaneous T cell lymphoma; circulatory diseases such asarteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritisnodosa and myocardosis; collagen diseases such as scleroderma, Wegener'sgranuloma and Sjogren's syndrome; adiposis; eosinophilic fasciitis;periodontal disease such as lesions of gingiva, periodontium, alveolarbone and substantia ossea dentis; nephrotic syndrome such asglomerulonephritis; male pattern aleopecia or alopecia senilis bypreventing epilation or providing hair germination and/or promoting hairgeneration and hair growth; muscular dystrophy; Pyoderma and Sezary'ssyndrome; Addison's disease; active oxygen-mediated diseases, as forexample organ injury such as ischemia-reperfusion injury of organs (suchas heart, liver, kidney and digestive tract) which occurs uponpreservation, transplantation or ischemic disease (for example,thrombosis and cardiac infarction); intestinal diseases such asendotoxin-shock, pseudomembranous colitis and colitis caused by drug orradiation; renal diseases such as ischemic acute renal insufficiency andchronic renal insufficiency; pulmonary diseases such as toxinosis causedby lung-oxygen or drug (for example, paracort and bleomycins), lungcancer and pulmonary emphysema; ocular diseases such as cataracta,siderosis, retinitis, pigmentosa, senile macular degeneration, vitrealscarring and corneal alkali burn; dermatitis such as erythemamultiforme, linear IgA ballous dermatitis and cement dermatitis; andothers such as gingivitis, periodontitis, sepsis, pancreatitis, diseasescaused by environmental pollution (for example, air pollution), aging,carcinogenesis, metastasis of carcinoma and hypobaropathy; diseasescaused by histamine or leukotriene-C4 release; Behcet's disease such asintestinal-, vasculo- or neuro-Behcet's disease, and also Behcet's whichaffects the oral cavity, skin, eye, vulva, articulation, epididymis,lung, kidney and so on. Furthermore, the compounds of the invention areuseful for the treatment and prevention of hepatic disease such asimmunogenic diseases (for example, chronic autoimmune liver diseasessuch as autoimmune hepatitis, primary biliary cirrhosis and sclerosingcholangitis), partial liver resection, acute liver necrosis (e.g.necrosis caused by toxin, viral hepatitis, shock or anoxia), B-virushepatitis, non-A/non-B hepatitis, cirrhosis (such as alcoholiccirrhosis) and hepatic failure such as fulminant hepatic failure,late-onset hepatic failure and “acute-on-chronic” liver failure (acuteliver failure on chronic liver diseases), and moreover are useful forvarious diseases because of their useful activity such as augmention ofchemotherapeutic effect, cytomegalovirus infection, particularly HCMVinfection, anti-inflammatory activity, sclerosing and fibrotic diseasessuch as nephrosis, scleroderma, pulmonary fibrosis, arteriosclerosis,congestive heart failure, ventricular hypertrophy, post-surgicaladhesions and scarring, stroke, myocardial infarction and injuryassociated with ischemia and reperfusion, and the like.

[0056] Additionally, compounds of the invention possess FK-506antagonistic properties. The compounds of the present invention may thusbe used in the treatment of immunodepression or a disorder involvingimmunodepression. Examples of disorders involving immunodepressioninclude AIDS, cancer, fungal infections, senile dementia, trauma(including wound healing, surgery and shock) chronic bacterialinfection, and certain central nervous system disorders. Theimmunodepression to be treated may be caused by an overdose of animmunosuppressive macrocyclic compound, for example derivatives of12-(2-cyclohexyl-1-methylvinyl)-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0 ^(4,9)]octacos-18-ene such as FK-506 or rapamycin. The overdosing of suchmedicants by patients is quite common upon their realizing that theyhave forgotten to take their medication at the prescribed time and canlead to serious side effects.

[0057] The ability of the compounds of the invention to treatproliferative diseases can be demonstrated according to the methodsdescribed in Bunchman E T and C A Brookshire, Transplantation Proceed.23 967-968 (1991); Yamagishi, et al., Biochem. Biophys. Res. Comm. 191840-846 (1993); and Shichiri, et al., J. Clin. Invest. 87 1867-1871(1991). Proliferative diseases include smooth muscle proliferation,systemic sclerosis, cirrhosis of the liver, adult respiratory distresssyndrome, idiopathic cardiomyopathy, lupus erythematosus, diabeticretinopathy or other retinopathies, psoriasis, scleroderma, prostatichyperplasia, cardiac hyperplasia, restenosis following arterial injuryor other pathologic stenosis of blood vessels. In addition, thesecompounds antagonize cellular responses to several growth factors, andtherefore possess antiangiogenic properties, making them useful agentsto control or reverse the growth of certain tumors, as well as fibroticdiseases of the lung, liver, and kidney.

[0058] Aqueous liquid compositions of the present invention areparticularly useful for the treatment and prevention of various diseasesof the eye such as autoimmune diseases (including, for example, conicalcornea, keratitis, dysophia epithelialis corneae, leukoma, Mooren'sulcer, sclevitis and Graves' ophthalmopathy) and rejection of cornealtransplantation.

[0059] When used in the above or other treatments, a therapeuticallyeffective amount of one of the compounds of the present invention may beemployed in pure form or, where such forms exist, in pharmaceuticallyacceptable salt, ester or prodrug form. Alternatively, the compound maybe administered as a pharmaceutical composition containing the compoundof interest in combination with one or more pharmaceutically acceptableexcipients. The phrase “therapeutically effective amount” of thecompound of the invention means a sufficient amount of the compound totreat disorders, at a reasonable benefit/risk ratio applicable to anymedical treatment. It will be understood, however, that the total dailyusage of the compounds and compositions of the present invention will bedecided by the attending physician within the scope of sound medicaljudgement. The specific therapeutically effective dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts. For example, it is wellwithin the skill of the art to start doses of the compound at levelslower than required to achieve the desired therapeutic effect and togradually increase the dosage until the desired effect is achieved.

[0060] The total daily dose of the compounds of this inventionadministered to a human or lower animal may range from about 0.01 toabout 10 mg/kg/day. For purposes of oral administration, more preferabledoses may be in the range of from about 0.001 to about 3 mg/kg/day. Forthe purposes of local delivery from a stent, the daily dose that apatient will receive depends on the length of the stent. For example, a15 mm coronary stent may contain a drug in an amount ranging from about1 to about 120 micrograms and may deliver that drug over a time periodranging from several hours to several weeks. If desired, the effectivedaily dose may be divided into multiple doses for purposes ofadministration; consequently, single dose compositions may contain suchamounts or submultiples thereof to make up the daily dose. Topicaladminstration may involve doses ranging from 0.001 to 3% mg/kg/day,depending on the site of application.

[0061] Pharmaceutical Compositions

[0062] The pharmaceutical compositions of the present invention comprisea compound of the invention and a pharmaceutically acceptable carrier orexcipient, which may be administered orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or transdermal patch), bucally, as an oral ornasal spray, or locally, as in a stent placed within the vasculature.The phrase “pharmaceutically acceptable carrier” means a non-toxicsolid, semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral,” as usedherein, refers to modes of administration which include intravenous,intraarterial, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection, infusion, and placement, suchas, for example, in vasculature.

[0063] Pharmaceutical compositions of this invention for parenteralinjection comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), carboxymethylcellulose and suitable mixturesthereof, vegetable oils (such as olive oil), and injectable organicesters such as ethyl oleate. Proper fluidity can be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

[0064] These compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents, and dispersingagents. Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents such as sugars, sodium chloride,and the like. Prolonged absorption of the injectable pharmaceutical formmay be brought about by the inclusion of agents that delay absorptionsuch as aluminum monostearate and gelatin.

[0065] In some cases, in order to prolong the effect of the drug, it isdesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

[0066] Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides) Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

[0067] The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium just prior to use.

[0068] Solid dosage forms for oral administration include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound is mixed with at least one inert, pharmaceuticallyacceptable excipient or carrier such as sodium citrate or dicalciumphosphate and/or a) fillers or extenders such as starches, lactose,sucrose, glucose, mannitol, and silicic acid, b) binders such as, forexample, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia, c) humectants such asglycerol, d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates, and sodiumcarbonate, e) solution retarding agents such as paraffin, f) absorptionaccelerators such as quaternary ammonium compounds, g) wetting agentssuch as, for example, cetyl alcohol and glycerol monostearate, h)absorbents such as kaolin and bentonite clay, and i) lubricants such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof. In the case of capsules,tablets and pills, the dosage form may also comprise buffering agents.

[0069] Solid compositions of a similar type may also be employed asfillers in soft, semi-solid and hard-filled gelatin capsules orliquid-filled capsules using such excipients as lactose or milk sugar aswell as high molecular weight polyethylene glycols and the like.

[0070] The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Those embedding compositionscontaining a drug can be placed on medical devices, such as stents,grafts, catheters, and balloons.

[0071] The active compounds can also be in micro-encapsulated form, ifappropriate, with one or more of the above-mentioned excipients.

[0072] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrupsand elixirs. In addition to the active compounds, the liquid dosageforms may contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethyl formamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof.

[0073] Besides inert diluents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and perfuming agents.

[0074] Suspensions, in addition to the active compounds, may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth,and mixtures thereof.

[0075] Topical administration includes administration to the skin ormucosa, including surfaces of the lung and eye. Compositions for topicaladministration, including those for inhalation, may be prepared as a drypowder which may be pressurized or non-pressurized. In non-pressurizedpowder compositions, the active ingredient in finely divided form may beused in admixture with a larger-sized pharmaceutically acceptable inertcarrier comprising particles having a size, for example, of up to 100micrometers in diameter. Suitable inert carriers include sugars such aslactose. Desirably, at least 95% by weight of the particles of theactive ingredient have an effective particle size in the range of 0.01to 10 micrometers. Compositions for topical use on the skin also includeoinments, creams, lotions, and gels.

[0076] Alternatively, the composition may be pressurized and contain acompressed gas, such as nitrogen or a liquified gas propellant. Theliquified propellant medium and indeed the total composition ispreferably such that the active ingredient does not dissolve therein toany substantial extent. The pressurized composition may also contain asurface active agent. The surface active agent may be a liquid or solidnon-ionic surface active agent or may be a solid anionic surface activeagent. It is preferred to use the solid anionic surface active agent inthe form of a sodium salt.

[0077] A further form of topical administration is to the eye, as forthe treatment of immune-mediated conditions of the eye such asautomimmue diseases, allergic or inflammatory conditions, and cornealtransplants. The compound of the invention is delivered in apharmaceutically acceptable ophthalmic vehicle, such that the compoundis maintained in contact with the ocular surface for a sufficient timeperiod to allow the compound to penetrate the corneal and internalregions of the eye, as for example the anterior chamber, posteriorchamber, vitreous body, aqueous humor, vitreous humor, cornea,iris/cilary, lens, choroid/retina and sclera. The pharmaceuticallyacceptable ophthalmic vehicle may, for example, be an ointment,vegetable oil or an encapsulating material.

[0078] Compositions for rectal or vaginal administration are preferablysuppositories or retention enemas which can be prepared by mixing thecompounds of this invention with suitable non-irritating excipients orcarriers such as cocoa butter, polyethylene glycol or a suppository waxwhich are solid at room temperature but liquid at body temperature andtherefore melt in the rectum or vaginal cavity and release the activecompound.

[0079] Compounds of the present invention can also be administered inthe form of liposomes. As is known in the art, liposomes are generallyderived from phospholipids or other lipid substances. Liposomes areformed by mono- or multi-lamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto a compound of the present invention, stabilizers, preservatives,excipients, and the like. The preferred lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art. See, for example,Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, NewYork, N.Y. (1976), p. 33 et seq.

[0080] Compounds of the present invention may also be coadministeredwith one or more immunosuppressant agents. The immunosuppressant agentswithin the scope of this invention include, but are not limited to,IMURAN® azathioprine sodium, brequinar sodium, SPANIDIN® gusperimustrihydrochloride (also known as deoxyspergualin), mizoribine (also knownas bredinin), CELLCEPT® mycophenolate mofetil, NEORAL® Cylosporin A(also marketed as different formulation of Cyclosporin A under thetrademark SANDIMMUNE®), PROGRAF® tacrolimus (also known as FK-506),sirolimus and RAPAMUNE®, leflunomide (also known as HWA-486),glucocorticoids, such as prednisolone and its derivatives, antibodytherapies such as orthoclone (OKT3) and Zenapax®, and antithymyocyteglobulins, such as thymoglobulins.

EXAMPLE 3

[0081] The purpose of this example was to determine the effects of arapamycin analog on neointimal formation in porcine coronary arteriescontaining stents. This example illustrates that the rapamycin analogA-1 79578, when compounded and delivered from the BiocompatiblesBiodiviYsio PC Coronary stent favorably affects neointimal hyperplasiaand lumen size in porcine coronary arteries. This finding suggests thatsuch a combination may be of substantial clinical benefit if properlyapplied in humans by limiting neointimal hyperplasia.

[0082] The agent A-179578 is a rapamycin analog. The study set forth inthis example was designed to assess the ability of the rapamycin analogA-179578 to reduce neointimal hyperplasia in a porcine coronary stentmodel. Efficacy of A-179578 in this model would suggest its clinicalpotential for the limitation and treatment of coronary restenosis instents following percutaneous revascularization. The domestic swine wasused because this model appears to yield results comparable to otherinvestigations seeking to limit neointimal hyperplasia in humansubjects.

[0083] The example tested A-179578 eluted from coronary stents placed injuvenile farm pigs, and compared these results with control stents. Thecontrol stents had polymer alone covering its struts. This is important,for the polymer itself must not stimulate neointimal hyperplasia to asubstantial degree. As the eluted drug disappears, an inflammatoryresponse to the polymer could conceivably result in a late “catch-upphenomenon” where the restenosis process is not stopped, but insteadslowed. This phenomenon would result in restenosis at late dates inhuman subjects.

[0084] Stents were implanted in two blood vessels in each pig. Pigs usedin this model were generally 2-4 months old and weighed 30-40 Kg. Twocoronary stents were thus implanted in each pig by visually assessing anormal stent:artery ratio of 1.1-1.2.

[0085] Beginning on the day of the procedure, pigs were given oralaspirin (325 mg daily) and continued for the remainder of their course.General anesthesia was achieved by means of intramuscular injectionfollowed by intravenous ketamine (30 mg/kg) and xylazine (3 mg/kg).Additional medication at the time of induction included atropine (1 mg)and flocillin (1 g) administered intramuscularly. During the stentingprocedure, an intraarterial bolus of 10,000 units of heparin wasadministered.

[0086] Arterial access was obtained by cutdown on the right externalcarotid and placement of an 8F sheath. After the procedure, the animalswere maintained on a normal diet without cholesterol or other specialsupplementation.

[0087] The BiodivYsio stent was used with nominal vessel target size of3.0 mm. See FIG. 2. Two coronary arteries per pig were assigned atrandom to deployment of the stents. The stent was either a drug elutingstent (polymer plus drug stent) or a stent coated with a polymer only(polymer only stent). The stents were delivered by means of standardguide catheters and wires. The stent balloons were inflated toappropriate sizes for less than 30 seconds.

[0088] Each pig had one polymer only stent and one polymer plus drugstent placed in separate coronary arteries, so that each pig would haveone stent for drug and one for control.

[0089] A sample size of 20 pigs total was chosen to detect a projecteddifference in neointimal thickness of 0.2 mm with a standard deviationof 0.15 mm, at a power of 0.95 and beta 0.02.

[0090] Animals were euthanized at 28 days for histopathologicexamination and quantification. Following removal of the heart from theperfusion pump system, the left atrial appendage was removed for accessto the proximal coronary arteries. Coronary arterial segments withinjuries were dissected free of the epicardium. Segments containinglesions was isolated, thereby allowing sufficient tissue to containuninvolved blood vessel at either end. The foregoing segments, eachroughly 2.5 cm in length, were embedded and processed by means ofstandard plastic embedding techniques. The tissues were subsequentlyprocessed and stained with hematoxylin-eosin and elastic-van Giesontechniques.

[0091] Low and high power light microscopy were used to make lengthmeasurements in the plane of microscopic view by means of a calibratedreticle and a digital microscopy system connected to a computeremploying calibrated analysis software.

[0092] The severity of vessel injury and the neointimal response weremeasured by calibrated digital microscopy. The importance of theintegrity of the internal elastic lamina is well-known to those skilledin the art. A histopathologic injury score in stented blood vessels hasbeen validated as being closely related to neointimal thickness. Thisscore is related to depth of injury and is as follows: Score Descriptionof Injury 0 Internal elastic lamina intact; endothelium typicallydenuded, media compressed but not lacerated. 1 Internal elastic laminalacerated; media typically compressed but not lacerated. 2 Internalelastic lacerated; media visibly lacerated; external elastic laminaintact but compressed. 3 External elastic lamina lacerated; typicallylarge lacerations of media extending through the external elasticlamina; coil wires sometimes residing in adventitia.

[0093] This quantitative measurement of injury was assessed for allstent wires of each stent section. The calibrated digital image was alsoused to measure at each stent wire site the neointimal thickness. Lumenarea, area contained with the internal elastic lamina, and area withinthe external elastic lamina were also measured.

[0094] At each stent wire site for a given section, the neointimalthickness was averaged to obtain a mean injury score for each section.The measurement of neointimal thickness was made to the abluminal sideof the stent wire, because the neointimal in all cases includes thisthickness.

[0095] The mid-stent segment was used for measurement, analysis, andcomparison. Data were also recorded (and included in the data section ofthis report) for proximal and distal segments.

[0096] The data analysis methods for this study did not need to takeinto account variable arterial injury across treatment/control groups,because mild to moderate injury is sensitive enough to detect treatmentdifferences. Paired t-testing was performed to compare variables acrossthe polymer only stents (control group) and polymer plus drug stents(treatment group). No animal died in this study before scheduledtimepoints.

[0097] Table 3 shows the pigs and arteries used. In Table 3, LCX meansthe circumflex branch of the left coronary artery, LAD means the leftanterior descending coronary artery, and RCA means the right coronaryartery. TABLE 3 Pigs and Vessels Used 1 2000-G-693 RCA - Control2000-G-693 LCX - Test 2 2000-G-698 RCA - Test 2000-G-698 LAD - Control 32000-G-702 RCA - Test 2000-G-702 LAD - Control 4 2000-G-709 RCA -Control 2000-G-709 LAD - Test 5 2000-G-306 RCA - Control 2000-G-306LAD - Test 2000-G-306 *LCX - Test 6 2000-G-672 RCA - Test 2000-G-672LAD - Control 7 2000-G-712 RCA - Control 2000-G-712 LCX - Test 82000-G-735 RCA - Control 2000-G-735 LAD - Test 9 2000-G-736 RCA -Control 2000-G-736 LCX - Test 10 2000-G-740 RCA - Test 2000-G-740 LAD -Control 11 2000-G-742 LAD - Test 2000-G-742 OM (LCX) - Control 122000-G-744 RCA - Test 2000-G-744 LAD - Control 13 2000-G-748 RCA - Test2000-G-748 LAD - Control 14 2000-G-749 RCA - Control 2000-G-749 LCX -Test 15 2000-G-753 RCA - Control 2000-G-753 LAD - Test 16 2000-G-754RCA - Test 2000-G-754 LCX - Control 17 2000-G-755 RCA - Control2000-G-755 LAD - Test 18 2000-G-756 RCA - Test 2000-G-756 LAD - Control19 2000-G-757 LAD - Control 2000-G-757 LCX - Test 20 2000-G-760 LAD -Test 2000-G-760 LCX - Control

[0098] Table 4 shows the summary results for all data for mean injuryand neointimal thickness for each stent, including proximal, mid, anddistal segments.

[0099] Table 4 also shows lumen size, percent stenosis, and artery sizeas measured by the internal elastic laminae (IEL) and external elasticlaminae (EEL). TABLE 4 Summary: All Measures (Distal, Mid, Proximal)mean % Neointimal ID prox ref dist ref lumen IEL EEL injury stenosisarea NIT Control Distal Mean 4.46 3.96 4.88 7.66 9.00 0.22 36.10 2.790.41 SD 1.20 1.16 1.30 1.15 1.10 0.26 15.41 1.29 0.17 Control Mid Mean4.46 3.96 4.94 7.71 9.08 0.08 36.23 2.77 0.38 SD 1.20 1.16 1.44 1.071.15 0.14 14.93 1.20 0.16 Control Proximal Mean 4.46 3.96 5.11 7.89 9.300.15 35.35 2.78 0.38 SD 1.20 1.16 1.38 1.33 1.42 0.22 11.94 1.04 0.12Test Distal Mean 4.26 3.41 6.04 7.70 9.01 0.26 22.35 1.66 0.25 SD 1.260.96 1.55 1.49 1.47 0.43  8.58 0.58 0.06 Test Mid Mean 4.26 3.41 6.357.75 8.98 0.04 18.71 1.41 0.22 SD 1.26 0.96 1.29 1.18 1.31 0.07  5.680.33 0.05 Test Proximal Mean 2.56 2.15 3.31 4.16 4.66 0.19 16.79 1.290.18 SD 1.66 1.37 2.39 3.48 4.15 0.13  9.97 0.80 0.12

[0100] There was no statistically significant difference for neointimalarea or thickness across proximal, mid, or distal segments within thetest group (polymer plus drug stents) or control groups (polymer onlystents). This observation is quite consistent with prior studies, andthus allows use of only the mid segment for statistical comparison oftest devices (polymer plus drug stents) vs. control devices (polymeronly stents).

[0101] Table 5 shows the statistical t-test comparisons across testgroups and control groups. There was a statistically significantdifference in neointimal thickness, neointimal area, lumen size, andpercent lumen stenosis, the drug eluting stent being clearly favored.Conversely, there were no statistically significant differences betweenthe test group (polymer plus drug stents) and the control group (polymeronly stents) for mean injury score, external elastic laminae, orinternal elastic laminae areas. TABLE 5 Statistical Comparison of Testvs. Control Parameters: Mid-Section Data t-test Statistics ParameterDifference t-test DF Std Error Lower 95% Upper 95% p Lumen −1.17 −2.2838 0.52 −2.21 −0.13 0.029 IEL 0.03 0.088 38 0.36 −0.71 0.78 0.93 EEL 0.20.499 38 0.39 −0.599 0.99 0.62 NI Thickness 0.18 5.153 38 0.034 0.1060.244 <.0001 NI Area 1.21 3.62 38 0.33 0.53 1.88 0.0008 Mean Injury0.038 1.137 38 0.033 −0.02 0.106 0.26 % Stenosis 14.54 2.97 38 4.9 4.6124.47 0.005

[0102] The reference arteries proximal and distal to the stentedsegments were observed, and quantitated. These vessels appeared normalin all cases, uninjured in both the control group (polymer only stents)and the test group (polymer plus drug stents). See FIGS. 3A and 3B. Thedata below show there were no statistically significant differences insize between the stents in the control group and the stents in the testgroup. Proximal Reference Distal Reference Diameter (mm) Diameter (mm)Control (mean ± SD) 4.46 ± 1.20 3.96 ± 1.16 Test (mean ± SD) 4.26 ± 1.263.41 ± 0.96

[0103] The data suggest that statistically significant differencesexist, and these differences favor the stent that elutes A-179578. Thestent of this invention results in lower neointimal area, lowerneointimal thickness, and greater lumen area. There were no significantdifferences within the test group (polymer plus drug stents) and thecontrol group (polymer only stents) for neointimal or injury parameters.There were no significant differences in artery sizes (including thestent) for the control group compared to the test group. These latterfindings suggest no significant difference in the arterial remodelingcharacteristics of the polymeric coating containing the drug.

[0104] At most, mild inflammation was found on both the polymer plusdrug stent and the polymer only stent. This finding suggests that thepolymer exhibits satisfactory biocompatibility, even without drugloading. Other studies show that when drug has completely gone from thepolymer, the polymer itself creates enough inflammation to causeneointima. This phenomenon may be responsible for the late catch-upphenomenon of clinical late restenosis. Because the polymer in thisexample did not cause inflammation in the coronary arteries, lateproblems related to the polymer after the drug is exhausted areunlikely.

[0105] In conclusion, a stent containing the compound A-179578 with apolymer showed a reduction in neointimal hyperplasia in the porcinemodel when placed in a coronary artery.

EXAMPLE 4

[0106] The purpose of this example is to determe the rate of release ofthe A-179578 drug from 316L Electropolished Stainless Steel Couponscoated with a a biocompatible polymer containing phosphorylcholine sidegroups.

[0107] Rubber septa from lids from HPLC vials were removed from thevials and placed into glass vials so that the “Teflon” side faced up.These septa served as supports for the test samples. The test sampleswere 316L stainless steel coupons that had been previously coated with abiocompatible polymer containing phosphorylcholine side groups (PCpolymer). Coronary stents are commonly made of 316L stainless steel andcan be coated with the PC polymer to provide a depot site for loadingdrugs. The coated coupons, which serve to simulate stents, were placedonto the septa. By using a glass Hamilton Syringe, a solution ofA-179578 and ethanol (10 μl) was applied to the surface of each coupon.The solution contained A-179578 (30.6 mg) dissolved in 100% ethanol (3.0ml). The syringe was cleaned with ethanol between each application. Thecap to the glass vial was placed on the vial loosely, thereby assuringproper ventilation. The coupon was allowed to dry for a minimum of 1.5hours. Twelve (12) coupons were loaded in this way—six being used todetermine the average amount of drug loaded onto the device and sixbeing used to measure the time needed to release the drug from thedevices.

[0108] To determine the total amount of ABT-578 loaded onto a coupon, acoupon was removed from the vial and placed into 50/50 acetonitrile/0.01M phosphate buffer (pH 6.0, 5.0 ml). The coupon was placed onto a 5210Branson sonicator for one hour. The coupon was then removed from thesolution, and the solution was assayed by HPLC.

[0109] The time release studies were performed by immersing and removingthe individual coupons from fresh aliquots (10.0 ml) of 0.01 M phosphatebuffer at a pH of 6.0 at each of the following time intervals—5, 15, 30and 60 minutes. For the remaining time points of 120, 180, 240, 300, 360minutes, volumes of 5.0 ml of buffer were used. To facilitate mixingduring the drug release phase, the samples were placed onto a Eberbachshaker set at low speed. All solution aliquots were assayed by HPLCafter the testing of the last sample was completed.

[0110] The HPLC analysis was performed with a Hewlett Packard series1100 instrument having the following settings: Injection Volume 100 μlAcquisition Time 40 minutes Flow Rate 1.0 ml/min Column Temperature 40°C. Wavelength 278 nm Mobile Phase 65% Acetonitrile/35% H₂O Column YMCODS-A S5 μm, 4.6 × 250 mm Part No. A12052546WT

[0111] The results from the above experiment showed the followingrelease data: TABLE 6 Time Percent Standard (min.) Release Deviation0.00 0.00 0.00 5.00 1.87 1.12 15.00 2.97 1.47 30.00 3.24 1.28 60.00 3.291.29 120.00 3.92 1.28 180.00 4.36 1.33 240.00 4.37 1.35 300.00 6.34 2.07360.00 7.88 1.01

EXAMPLE 5

[0112] The purpose of this example was to determine the loading andrelease of ABT-578 from 15 mm BiodivYsio drug delivery stents.

[0113] To load the stents with drug, a solution of ABT 578 in ethanol ata concentration of 50 mg/ml was prepared and dispensed into twelvevials. Twelve individual polymer-coated stents were placed on fixturesdesigned to hold the stent in a vertical position and the stents wereimmersed vertically in the drug solution for five minutes. The stentsand fixtures were removed from the vials and excess drug solution wasblotted away by contacting the stents with an absorbant material. Thestents were then allowed to dry in air for 30 minutes in an invertedvertical position.

[0114] The stents were removed from the fixtures, and each stent wasplaced into 50/50 acetonitrile/phosphate buffer (pH 5.1, 2.0 ml) andsonicated for one hour. The stents were removed from the solution andsolutions were assayed for concentration of drug, which allowedcalculation of the amount of drug originally on the stents. This methodwas independently shown to remove at least 95% of the drug from thestent coating. On average, the stents contained 60 micrograms of drug±20micrograms.

[0115] The drug-loaded stents were placed on the fixtures and placedinto 0.01 M phosphate buffer (pH=6.0,1.9 ml) in individual vials. Thesesamples were placed onto a Eberbach shaker set at low speed to provideback-and-forth agitation. To avoid approaching drug saturation in thebuffer, the stents were transferred periodically to fresh buffer vialsat the following points: 15, 30, 45, 60, 120,135, 150,165, 180, 240, 390minutes. The dissolution buffer vials were assayed by HPLC for the drugconcentration at the end of the drug release period studied. The data,represented as % cumulative release of the drug as a function of time,is shown in tabular form below: TABLE 7 Time (min) % Cumulative Releaseof Drug 15 0.3 30 1.1 45 2.1 60 3.2 120 4.3 135 5.9 150 5.3 165 6.8 1807.4 240 10.8 390 13.2

[0116] It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents. Various changes andmodifications to the disclosed embodiments will be apparent to thoseskilled in the art. Such changes and modifications, including withoutlimitation those relating to the chemical structures, substituents,derivatives, intermediates, syntheses, formulations and/or methods ofuse of the invention, may be made without departing from the spirit andscope thereof.

What is claimed is:
 1. A medical device comprising a supportingstructure having a coating on the surface thereof, said coatingcontaining the therapeutic substance

or a pharmaceutically acceptable salt or prodrug thereof.
 2. The medicaldevice of claim 1, wherein said supporting structure is selected fromthe group consisting of coronary stents, peripheral stents, catheters,arterio-venous grafts, by-pass grafts, and drug delivery balloons usedin the vasculature.
 3. The medical device of claim 1, wherein saidcoating is polymeric.
 4. The medical device of claim 3, wherein saidpolymeric coati ng is biostable.
 5. The medical device of claim 3,wherein said polymeric coating is biodegradable.
 6. The medical deviceof claim 1, wherein said therapeutic substance is

or a pharmaceutically acceptable salt or prodrug thereof.
 7. The medicaldevice of claim 1, wherein said therapeutic substance is

or a pharmaceutically acceptable salt or prodrug thereof.
 8. A medicaldevice comprising a supporting structure having a coating on the surfacethereof, said coating containing the therapeutic substance

or a pharmaceutically acceptable salt or prodrug thereof.
 9. The medicaldevice of claim 8, wherein said supporting structure is selected fromthe group consisting of coronary stents, peripheral stents, catheters,arterio-venous grafts, by-pass grafts, and drug delivery balloons usedin the vasculature.
 10. The medical device of claim 8, wherein saidcoating is polymeric.
 11. The medical device of claim 10, wherein saidpolymeric coating is biostable.
 12. The medical device of claim 10,wherein said polymeric coating is biodegradable.
 13. A medical devicecomprising a supporting structure having a coating on the surfacethereof, said coating containing the therapeutic substance

or a pharmaceutically acceptable salt or prodrug thereof.
 14. Themedical device of claim 13, wherein said supporting structure isselected from the group consisting of coronary stents, peripheralstents, catheters, arterio-venous grafts, by-pass grafts, and drugdelivery balloons used in the vasculature.
 15. The medical device ofclaim 13, wherein said coating is polymeric.
 16. The medical device ofclaim 15, wherein said polymeric coating is biostable.
 17. The medicaldevice of claim 15, wherein said polymeric coating is biodegradable.